Improvement and establishment of the tTA-dependent inducible system in the mouse brain

Citation of documents: Please do not cite the URL that is displayed in your browser location input, instead use the DOI, URN or the persistent URL below, as we can guarantee their long-time accessibility.

Translation of abstract (English)

A genetic regulatory system employing the tTA that can induce individual gene expression reversibly in a temporal and spatial manner could permit a more precise analysis of gene functions on animal physiology and behaviour. For efficient expression of tTA in the mouse, the coding sequence of the htTA was altered concerning mammalian codon usage, putative splicing signals, CG dinucleotide content, and toxicity of the transactivation domain VP16. These sequence changes on tTA resulted in the three-fold increase of the inducible activity in transiently transfected HeLa cells, compared to the prokaryotic one. Activity of the htTA was efficiently suppressed by addition of doxycycline. To express the htTA in a neuron- and region-specific manner in mouse brain, the region-specific promoter of the KA1 was chosen. The htTA gene under the KA1 promoter appears to be expressed too low to induce tTA responsive genes (e.g. lacZ) in these transgenic mice. An alternative approach for region specific htTA expression was to use the CaMKIIa promoter but to repress the forebrain-specific promoter in regions other than hippocampus by the neuron-restrictive silencer element (NRSE) of the NR2C subunit. The expression pattern and activity of the htTA under control of the NR2C silencer-containing chimeric promoter were observed restrictively in hippocampus of two lines, namely only in DG, and in CA1 and DG, respectively. Activity of the htTA was detected at postnatal stage and was turned off completely by doxycycline. It is demonstrated here that the htTA provides efficient expression and reversible inducibility in vitro and in vivo and that the NR2C silencer-containing chimeric promoter was successfully applied in transgenic mice. The hippocampus-specific genetic switch by the htTA should make it possible to study genes of interest involved in hippocampal functions and should advance the research of gene function in the CNS (central nervous system).